Subject of this invention are DNA constructs encoding fusion proteins comprising amino acid sequences of blood coagulation factors and of P-Selectin.
P-Selectin is an integral transmembrane glycoprotein expressed in endothelial cells and platelets. P-Selectin is an inducible molecule implicated in cell-to-cell adhesion. The molecule is stored on resting cells in particular sub-cellular compartments: Weibel-Palade bodies in endothelial cells and a-granules in platelets. The intracellular domain of P-Selectin (psel) was shown to be responsible for the targeting of the molecule in different compartments: storage granules, lysosomes, dense-core granules and synaptic-like-microvesicles [2, 4, 5]. Two chimeric molecules were generated using this domain: Tissue-Factor-psel and Horse-Radish Peroxidase (HRP)-psel. In both cases, using secretion-regulated cell lines (AtT20 and PC12), the chimeric molecules were directed into secretory granules and lysosomes [2, 6]. The intracellular tail possesses two distinct stretches of amino acids implicated in the targeting. The 10 amino acids close to the plasma membrane direct P-Selectin and HRP-psel in lysosomes, whereas the 3xe2x80x2extremity is required for the targeting in synaptic-like microvesicles, i.e., secretory vesicles [1, 6]. The targeting potential of the cytoplasmic tail was also shown to be increased in the presence of the transmembrane domain. When both domains were present, a chimeric E-selectin-P-selectin was more efficiently directed into granules [3].
The addition of the P-Selectin tail seems to represent an interesting tool for targeting genetic factors of therapeutic interest into intracellular storage compartments where they can be useful for somatic gene therapy. The present invention shows that DNA constructs of blood clotting factors like Factor IX and of P-Selectin create new possibilities for the treatment of patients suffering from a defiency of blood coagulation factors. It is directed to chimeric molecules comprising inter alia FIX-DNA constructs fused with the cytoplasmic domain of P-Selectin.
It has been found that a DNA construct encoding a fusion protein comprising an amino acid sequence of a blood clotting factor and an amino acid sequence of the cytoplasmic domain of human P-Selectin retains the full blood coagulation activity and has valuable properties which are useful for the somatic gene therapy of patients suffering from a defiency of a blood coagulation factor like Factor IX.